Specific binding sites in collagen for integrins and use thereof

ABSTRACT

The present invention identified a high affinity binding sequence in collagen type III for the collagen-binding integrin I domains. Provided herein are the methods used to characterize the sequence, the peptides comprising this novel sequence and the use of the peptides in enabling cell adhesion. Also provided herein are methods to identify specific integrin inhibitors, sequences of these inhibitors and their use in inhibiting pathophysiological conditions that may arise due to integrin-collagen interaction.

CROSS REFERENCE TO RELATED APPLICATION

This non-provisional application claims benefit of provisionalapplication U.S. Ser. No. 60/687,432 filed on Jun. 3, 2005, nowabandoned.

FEDERAL FUNDING LEGEND

This invention was produced using funds obtained through grant AR44415from the National Institutes of Health. Consequently, the federalgovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of computer-aidedmolecular modeling and interaction of extracellular matrix protein withreceptors and cell signaling. More specifically, the present inventionrelates to identification of motifs within the extracellular matrixprotein, collagen type III that binds I domains of the integrins anddesigning of specific inhibitors that inhibit the interaction of the Idomain of integrin with the collagen.

2. Description of the Related Art

Collagen is a major component of the extracellular matrix (ECM). Atleast 27 genetically different collagen types have been identified, eachcontaining at least one dominant collagenous domain (Ramchandran, G. N.,1988). These collagenous domains have a characteristic triple helixstructure formed by repeating Gly-X-Y sequences in each participatingchain where X often is Proline and Y is hydroxyproline. The collagenmonomers often assemble into more complex structures of varyingorganizations such as fibrils (types I-III, V and XI), networks (typesIV, VIII and X) and beaded filaments (type VI) (Hulmes, D. J., 1992).The fibrillar collagen types I and III are the major structuralcomponents of the ECM of skin, cardiac and vascular tissues, whereastype II collagen is a major structural component of cartilage. Inaddition to contributing to the structural integrity of the tissues,collagens also affect cell behaviour through interactions with othermatrix proteins and cellular receptors (Prockop, D. J. and Kivirikko,1995; Kuivaniemi, H., et al., 1997; Gelse, K. et. al., 2003; Myllyharju,J. and Kivirikko, K. I., 2001).

The integrins are a family of heterodimeric cell surface receptorsinvolved in cell-cell and cell-substrate adhesion. They act as bridgingmolecules that link intracellular signaling molecules to the ECM throughbi-directional signaling and control cell behaviour and tissuearchitecture (Hynes, R. O., 1992). Four integrins, α₁β₁, α₂β₁, α₁₀β₁ andα₁₁β₁ have been shown to bind collagens (Kramer, R. H. and Marks, N.,1989; Camper, L. et al., 1998; Velling, T. et al., 1999). Of these, theα₁β₁ and α₂β₁ integrins have been studied In more detail compared to theothers. Collagen integrin interactions play a role in normal andpathological physiology and directly affect cell adhesion, migration,proliferation and differentiation as well as angiogenesis, plateletaggregation and ECM assembly (Gulberg and Lundgren-Akerlund, 2002).However, the precise molecular mechanisms that lead to these activitiesare not understood.

Collagen binding by the four integrins is mediated by a ˜200 amino acidslong so-called inserted domain (I domain) found between blades 2 and 3of the β-propeller domain of the α chains. All four I domains (α₁I, α₂I,α₁₀I, α₁₁I) contain a metal ion-dependent adhesion site (MIDAS) that isrequired for coordinating a divalent cation and is essential forcollagen binding. Synthetic collagen peptides containing the type Icollagen derived sequences, GFOGER (SEQ ID NO: 1) or GLOGER (SEQ ID NO:2) have been reported to bind with high affinity to α₁I, α₂I and α₁₁I;furthermore, synthetic peptides containing these sequences inhibit thebinding of I domains to intact collagens (Knight, C. G. et al., 1998;Zhang, W. M. et al., 2003; Siljander, P. R. et al., 2004). The crystalstructures of apo-α₂I and α₂I in complex with a collagen peptidecontaining the GFOGER (SEQ ID NO: 1) sequence have been solved (Emsley,J. et al., 2000) and showed that the apo-α₂I adopted an inactive“closed” conformation and the ligand bound α₂I, an active “open”conformation (Lee, J. O. et al., 1995). The Glu residue in the collagenpeptide was shown in the structure of the complex to directly interactwith a Mg²⁺ ion coordinated by the MIDAS motif and the Arg residue formsa salt bridge with D₂₁₉ in α₂I. The importance of the GER sequence incollagen for integrin binding was confirmed by mutagenesis studies,which showed that replacing Glu in the collagen peptide with an Aspresidue completely abolished the binding whereas replacing the Arg witha Lys residue reduced the binding by 50% (Knight, C. G. et al., 2000).The Phe residue in the collagen sequence appeared to participate inhydrophobic interactions with α₂I and could be replaced by Leu. BothGFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2) bind to α₁I and α₂I (Xu,Y. et al., 2000). However, changing the Phe residue to a Met or an Alareduced the apparent affinity of I domains (Siljander, P. R. et al.,2004). GASGER (SEQ ID NO: 3) was also reported to be recognized by the Idomains but bound with lower affinity than GFOGER (SEQ ID NO: 1) andGLOGER (SEQ ID NO: 2) (Zhang, W. M. et al., 2003; Siljander, P. R. etal., 2004; Xu, Y. et al., 2000). Therefore, GFOGER (SEQ ID NO: 1) andGLOGER (SEQ ID NO: 2) are the only two known collagen-derived sequencemotifs that support high affinity binding by the collagen-binding Idomains. However, the GFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2)motifs are absent in some collagens such as human type III collagen.Additionally, previous studies have shown that CHO cell expressing α₁β₁and α₂β₁ could adhere and spread on human type III collagen andfurthermore, the recombinant proteins of α₁I and α₂I could bind to thiscollagen type (Nykvist, P. et al., 2000)

Thus, there is a need in the art for understanding the mechanism bywhich type III collagen binds to integrins and in designing of integrinspecific inhibitors. The present invention fulfills this long-standingneed and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a binding motif for collagenbinding I domains comprising amino acid sequence GROGER (SEQ ID NO: 4).The present invention is further directed to a method of identifying aninhibitor of integrin-collagen interaction. Such a method comprisesdesigning a test compound comprising a sequence that is specificallyrecognized by the I domain of the integrin, wherein said design is basedon computer-aided molecular modeling. Further, the level of binding ofthe integrin to the collagen is compared in the presence or absence ofthe test compound, where a decrease in binding of the integrin to thecollagen in presence of the test compound indicates that the testcompound is the inhibitor of the integrin-collagen interaction.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the analyses of the binding of α₁I and α₂I to fibrillarcollagen type I-III by SPR. Representative profiles of the relative SPRresponses of the binding of 1 μM α₁I and α₂I to immobilized type Iprocollagen is shown in FIG. 1A, type II mature collagen is shown inFIG. 1B and type III procollagen is shown in FIG. 1C in the presence of1 mM MgCl2. Responses to blank cells were subtracted. Y axis values areRUs normalized to the maximum RUs of a1I binding to type I-IIIcollagens, respectively

FIG. 2 shows electron micrographs of human type III procollagen incomplex with α₁I or α₂I following rotary shadowing. The C-terminalpropeptide of type III collagen, indicated by arrowheads, appeared as aknob at one end of each of the collagen molecules. Bound α₁I and α₂I,indicated by arrows, appeared as beads along the collagen strands. Thescale bars indicate 100 nm.

FIG. 3 shows histograms of the binding events of α₁I and α₂I along humantype III procollagen. The events were binned every 10 nm, and thepercentages of the binding events in each 10 nm bin over the totalbinding events counted are given. For α₁I, a total of 269 binding eventswere counted, and for α₂I, a total of 299 events were counted.

FIGS. 4A-C show collagen peptides from putative high affinity bindingsequences in type III collagen. FIG. 4A shows the amino acid sequence(SEQ ID NO: 18) of the high affinity binding region corresponding to270-300 nm from the C-terminal propeptide, indicated by underlinedletters, and its flanking area in human type III collagen. Residuesaround the GER motif are indicated by upper case letters. FIG. 4B showsthe amino acid sequences of the synthetic collagen peptides. Human typeIII collagen specific sequences are indicated by underlined and uppercase letters, and type I collagen specific sequences are indicated byupper case letters. Three GPO triplets are included on either side toensure the formation of triple helices. FIG. 4C shows circular dichroismspectra of the synthetic collagen peptides. Peptides #1, #2 and #3 inFIGS. 4B and 4C are identified as SEQ ID NO: 15, 16 and 17,respectively.

FIGS. 5A-D show inhibition of the binding of α₁I and α₂I to collagens bysynthetic collagen peptides. Different concentrations of peptides(0.01-100 μM) were mixed with 0.5 μM α₁I (FIGS. 5A, 5C) and 5 μM α₂I(FIGS. 5B, 5D) before being added to microtiter wells coated with typeIII procollagen (FIGS. 5A, 5B) or type I collagen (FIGS. 5C, 5D). Boundα₁I or α₂I were detected by anti-His monoclonal antibody, followed bygoat anti-mouse IgG (H+L)-alkaline phosphatase conjugate. The binding inthe absence of peptide was set to 100%. Data were presented as the meanvalue±S.E. of A405 nm (n=3) from a representative experiment. Peptides#1, #2 and #3 in these figures are identified as SEQ ID NO: 15, 16 and17, respectively.

FIGS. 6A-C show representative SPR sensorgrams of α₁I and α₂I binding toimmobilized peptide #1 (SEQ ID NO: 15). Increasing concentrations (0.5,1, 3, 6, 10 and 30 nM) of α₁I (FIG. 6A) or α₂I (FIG. 6B), were passedover a surface containing synthetic peptide #1 (SEQ ID NO: 15).Responses to blank cells were subtracted. The binding was abolished inthe presence of 2 mM EDTA (FIG. 6C).

FIG. 7 shows adhesion of MRC-5 cells to synthetic collagen peptides.Microtiter wells were coated with increasing concentrations of type IIIcollagen or collagen peptides (SEQ ID NO: 15 or SEQ ID NO: 16). Bothpeptides were present in a triple helix conformation and immobilized tosimilar extent in the wells as tested by the amount of recombinant CANbound to the two substrates (data not shown). Approximately 1.5×10⁴cells were added to the wells and allowed to adhere to the substrates atroom temperature for 45 min. Bound cells were fixed and then detectedwith crystal violet.

FIGS. 8A-D show analyses of GROGER (SEQ ID NO: 4) as the minimal bindingsequence for α₁I and α₂I. FIG. 8A shows the amino acid sequences of thesynthetic collagen peptides (SEQ ID NOs: 19, 20, 21, 15). Three GPOtriplets are included on either side to ensure the formation of triplehelices. FIG. 8B shows temperature-dependent denaturation profiles ofsynthetic collagen peptides. Ellipticity at 225 nm was measured astemperature increased from 10 to 50° C. at the rate of 20° C./hr.Melting points of peptides were calculated by nonlinear fitting usingthe Boltzmann sigmoidal equation (GraphPad Prism). FIGS. 8C and 8D showinhibition of the binding of α₁I and α₂I to type III collagen bysynthetic collagen peptides. Different concentrations of peptides weremixed with 0.5 μM α₁I (FIG. 8C) and 5 μM α₂I (FIG. 8D) before beingadded to microtiter wells coated with human mature type III collagen(FibroGen). The binding in the absence of peptide was set to 100%. Datawere presented as the mean value±S.E. of A405 nm (n=3) from arepresentative experiment.

FIGS. 9A-D show computer modeling of the interactions between α₂I andsynthetic collagen peptide #1 (SEQ ID NO: 15). The trailing and middlestrands of the collagen peptide and the backbones of α₂I are presentedin green, yellow, and grey, respectively. All residues displayed areshown with oxygen in red, nitrogen in blue and carbon in white, green oryellow for residues from α₂I, the trailing and the middle strand ofcollagen, respectively. The interactions between Asn¹⁵⁴ and Gln²¹⁵ ofα₂I and the middle strand of the collagen peptide GFOGER (SEQ ID NO: 1)(FIG. 9A) and GROGER (SEQ ID NO: 4) (FIG. 9B), and Leu²⁸⁶ and Tyr¹⁵⁷ ofα₂I and the trailing strand of GFOGER (SEQ ID NO: 1) (FIG. 9C) andGROGER (SEQ ID NO: 4) (FIG. 9D) are shown.

FIGS. 10A-D are computer modeling profiles showing specificity ofcollagen sequence to different I domains of collagen binding integrinalpha subunits. Computational prediction of I domain-collagen peptidecomplex used X-ray crystal structure of α₂I domain with collagenpeptide, GFOGER (SEQ ID NO: 1) as template. FIG. 10A shows the alpha-1 Idomain in complex with collagen peptide. FIG. 10B shows the alpha-2 Idomain in complex with collagen peptide. FIG. 10C shows alpha-10 Idomain in complex with collagen peptide. FIG. 10D shows alpha-11 Idomain in complex with collagen peptide.

The binding affinities is shown as follows: + represents bindingaffinity close to the template (blue), ++ represents slightly higherbinding affinity (green), +++ represents much higher binding affinity(dark green), − represents lower binding affinity (yellow) and −−represents disrupted binding affinity (red). In the computationalmutation in a collagen peptide, F8 on middle and trailing strand weresubstituted simultaneously, E11 on middle strand was replaced for eachmutation simulation and R12 on middle and trailing strand weresubstituted simultaneously for each mutation simulation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention identified a novel sequence motif, GROGER (SEQ IDNO: 4) from human type III collagen and characterized its binding to theI domains of integrins α₁and α₂. Briefly, the binding of recombinant Idomains from integrins α₁and α₂ (α₁I and α₂I) to fibrillar collagenstypes I-III was characterized and it was observed that each I domainbound to the three types of collagen with similar affinities. Usingrotary shadowing followed by electron microscopy, a high affinitybinding region was identified in the human type III collagen that wasrecognized by α₁I and α₂I. Examination of the region further revealedthe presence of two sequences that contained the critical GER motifs,GROGER (SEQ ID NO: 4) and GAOGER (SEQ ID NO: 5). Synthetic collagen-likepeptides containing these two motifs were synthesized and their triplehelical nature was confirmed by circular dichroism spectroscopy.

Further, it was demonstrated that the GROGER (SEQ ID NO: 4)-containingpeptides were able to bind to both α₁I and α₂I with high affinity andeffectively inhibit the binding of α₁I and α₂I to type III and type Icollagens, whereas the GAOGER (SEQ ID NO: 5)-containing peptide wasconsiderably less effective. Furthermore, the GROGER (SEQ ID NO:4)-containing peptide supported adhesion of human lung fibroblast cellswhen coated on a culture dish. Additionally, the present invention alsodisclosed the use of computer-aided molecular modeling to identifysequences that are specifically recognized by individual I domains onthe integrins. Thus, the finding of the present invention helps tounderstand the molecular interactions between collagens and integrins.It is further contemplated to synthesize peptides comprising thespecific sequences identified by the modeling approach and test theirutility in inhibiting integrin-collagen interaction and affect thebiological and pathological conditions that arise due to suchinteraction.

Type III collagen is a homotrimeric molecule and is a member of thefibrillar collagen family. It co-localizes with type I collagen intissues such as blood vessels and skin and plays a role in thedevelopment of these tissues (Prockop, D. J and Kivirikko, K. I., 1995;Liu, X et al., 1997). In vitro, it has been reported that the type IIIcollagen was able to support adhesion and spreading of cells expressingintegrin α₁β₁ or α₂β₁ (Nykvist, P. et al., 2000). However, human typeIII collagen does not contain the two previously known high affinityintegrin-binding motifs, GFOGER (SEQ ID NO: 1) and GLOGER (SEQ Id NO:2). The present invention compared the binding of α₁I and α₂I to thethree different types of fibrillar collagen (types I, II and III).Surface plasmon resonance (SPR) analysis showed that all three collagentypes contained at least two classes of binding sies for the two Idomains. A high affinity integrin-binding site was located by rotaryshadowing of I domains in complex with type III procollagen and asynthetic collagen triple helix peptide containing the GROGER (SEQ IDNO: 4) sequence was shown to bind with high affinity to the I domainsand could serve as a substrate for integrin-dependent cell adhesion.Other I domain-binding sites in type III collagen indicated by therotary shadowing experiment might represent low affinity sites.

Recently, the role of hydrophobic residues at the second position inGFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2) sequences was studied,and the interactions between α₂I and a number of GER-containing collagenpeptides comprising 6 amino acids with Glycine as the first amino acidand differing only in the second or the third position from GLOGER (SEQID NO: 2) or GFOGER (SEQ ID NO: 1) was examined (Siljander et al.,2004). It was observed that with respect to the second position, theorder of the inhibition potency was F≧L≧M>A. All these residues werehydrophobic or non-polar. However, the GROGER (SEQ ID NO: 4) sequenceidentified in this invention contains a charged residue at the secondposition. Furthermore, using the peptide inhibition assay to compare theapparent affinity of GROGER (SEQ ID NO: 4) to integrin I domains withthat of GFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2), the presentinvention demonstrated that GROGER exhibited a somewhat higher affinitythan GFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2) for α₂I andslightly lower affinity for α₁I. These observation might suggest thatdifferent integrins recognise different sites in collagen with differentaffinities.

Furthermore, to investigate how the presence of a charged residue wouldaffect the interactions with the I domains, computer modeling wasperformed based on the published structure of α₂I in complex of aGFOGER(SEQ ID NO: 1)-containing collagen peptide. Interestingly, thechange from Phe (F) to Arg (R) did not affect the positions ofneighboring amino acid residues in α₂I. An additional hydrogen bondinteraction was observed between the Arg (R) residue and the carbonylbackbone of Gln²¹⁵ in α₂I. This additional contact might explain theslightly higher observed affinity of α₂I for GROGER (SEQ ID NO: 4)compared with GLOGER (SEQ ID NO: 2). Whether this change affected thedownstream signaling events by integrins was not clear.

A search of different collagen sequences for the presence of GROGER (SEQID NO: 4) indicated that it was present in a variety of collagen types(Table 1). Noticeably, it was present in all the type I collagensexamined, yet, this sequence was not identified as a high affinitybinding sequence in previous studies with type I collagen (Xu, Y. etal., 2000). A more detailed examination revealed that it was onlypresent in the α₂I chain of type I collagens from bovine or chicken,which were the sources of type I collagens in the previous studies. Astype I collagen is composed of two α₁ chains and one α₂ chain, thepresence of GROGER (SEQ ID NO: 4) in the α₂ chain might not providesufficient interactions with residues in the I domains to allow a highaffinity binding. However, it would be interesting to examine whetherthis motif mediates a high affinity interactions between thecollagen-binding I domains and type of collagen from human, mouse or dogis interesting.

TABLE 1 The presence of GROGER (SEQ ID NO: 4) sequence in differenttypes of collagen. Position of the Species Type of collagen α chainstarting Gly Human Type I α₁ (I) 239 α₂ (I) 151 Type III α₁ (III) 237Type VII α₁ (VII) 2055 Type X α₁ (X) 197 Mouse Type I α₁ (I) 228 α₂ (I)157 Type III α₁ (III) 236 Type X α₁ (X) 197 Dog Type I α₁ (I) 235 α₂ (I)151 Chicken Type I α₂ (I) 150 Type XIV α₁ (XIV) 1697 Rat Type I α₂ (I)157 Bovine Type I α₂ (I) 149 Bullfrog Type I α₂ (I) 142

The present invention also contemplates using a computer-aided molecularmodeling approach to identify sequences that are specifically recognizedby individual I domains on the integrins and thus can be used as basefor developing integrin specific inhibitors. Such sequences can also beused to design specific binding sites in recombinant collagen orcollagen-like proteins and to synthesize peptides that could be used asinhibitors of integrin-collagen interaction.

In one embodiment of the present invention, there is provided a bindingmotif for collagen-binding I domains comprising an amino acid sequenceGROGER (SEQ ID NO: 4). The I domains of integrins bound by such a motifinclude but may not be limited to α₁and α₂ integrins. Moreover, themotif may comprise a charged amino acid in second position. Generally,the charged amino acid may form a hydrogen bond with carbonyl backgroundof gluatmine at position 215 in α₂I. Specifically, the charged aminoacid may be arginine. Further, the motif may be present on N-terminal ofhuman type III collagen or in the α₁ and α₂ chain of human type Icollagen.

In a related embodiment of the present invention, there is provided arecombinant collagen or collagen like protein comprising the bindingmotif described supra. Such a recombinant collagen or collagen-likeprotein may have sequence of SEQ ID NO: 15 or SEQ ID NO: 21. In afurther related embodiment of the present invention, there is providedan expression vector. Such an expression vector may comprise a DNAsequence encoding the recombinant collagen or collagen-like proteindescribed earlier. In a still further related embodiment of the presentinvention, there is provided a host cell comprising and expressing theexpression vector described earlier.

In another embodiment of the present invention, there is provided asynthetic collagen or collagen-like peptide comprising the binding motifdescribed supra. Although not limited to, such a peptide may havesequence of SEQ ID NO: 15 or SEQ ID NO: 21. Generally, such a peptidehas a triple helical structure. Further, such a synthetic peptide maybind I domains of integrins α₁ and α₂. In yet another embodiment of thepresent invention, there is provided a method of identifying aninhibitor of an integrin-collagen interaction, comprising: designing atest compound comprising a sequence that is specifically recognized bythe I domain of the integrin, where the design is based oncomputer-aided molecular modeling, and comparing the level of binding ofthe integrin to the collagen in the presence or absence of the testcompound, where a decrease in binding of the integrin to the collagen inpresence of the test compound indicates that the test compound is theinhibitor of the integrin-collagen interaction. The examples of integrinthat are bound by such inhibitors may not be limited to but includeα₁β₁, α₂β₁, α₁₀β₁ or α₁₁β₁. The collagen whose interaction with theintegrin is affected may be a type I, II or III collagen. Further, theinhibitor has a triple helical structure and may comprise an amino acidsequence including but not limited to GFPGER (SEQ ID NO: 6), GFOGEN (SEQID NO: 7), GFOGEK (SEQ ID NO: 8), GNOGER (SEQ ID NO: 9), GSOGER (SEQ IDNO: 10), GVOGER (SEQ ID NO: 11) or GPOGER (SEQ ID NO: 12).

In a related embodiment of the present invention, there is provided aninhibitory compound that is identified by the method described above. Ina further related embodiment of the present invention, there is provideda recombinant collagen or collagen-like protein comprising theinhibitory sequence indentified by the method described supra. In astill further related embodiment of the present invention, there isprovided an expression vector, comprising a DNA sequence encoding therecombinant collagen or collagen-like protein described supra. Inanother related embodiment of the present invention, there is provided ahost cell comprising and expressing the expression vector describedearlier.

In a further related embodiment of the present invention, there isprovided a synthetic collagen or collagen-like peptide comprising theinhibitory sequence identified by the method described supra. Further,the inhibitory sequence in a triple helical structure may not be limitedto but includes GFPGER (SEQ ID NO: 6), GFOGEN (SEQ ID NO; 7), GFOGEK(SEQ ID NO: 8), GNOGER (SEQ ID NO: 9), GSOGER (SEQ ID NO: 10), GVOGER(SEQ ID NO: 11) or GPOGER (SEQ ID NO: 12). Examples of integrins thatare bound by such peptides are the same as discussed earlier.

In another embodiment of the present invention, there is provided amethod of inhibiting integrin-collagen interaction, comprising:contacting a sample comprising the integrin and the collagen with thepeptide discussed supra, where the peptide binds the integrin with agreater affinity than the collagen, thereby inhibiting theintegrin-collagen interaction. Examples of biological processescontributed by integrin-collagen interaction and may be affected by thepeptide may not be limited to but include cell adhesion, cell migration,cell proliferation, cell differentiation, angiogenesis, plateletaggregation or extracellular matrix assembly. In a still further relatedembodiment of the present invention, there is provided a pharmaceuticalcomposition comprising the inhibitory compound identified by the methoddescribed supra and a pharmaceutically acceptable carrier.

In yet another embodiment of the present invention, there is provided amethod of treating an individual having a pathophysiological conditionresulting from integrin-collagen interaction, comprising the step ofadministering to the individual a pharmacologically effective amount ofthe composition discussed supra, such that the composition inhibits theintegrin-collagen interaction, thereby treating the individual havingthe pathophysiological condition. Examples of the pathophysiologicalconditions are may not be limited to but include inflammation, tumorgrowth, metastasis or angiogenesis.

As used herein, the term “compound” or “inhibitor” or “inhibitorycompound” means a molecular entity of natural, semi-synthetic orsynthetic origin that blocks, stops, inhibits, and/or suppressesintegrin interactions with collagen.

As used herein, the term “contacting” refers to any suitable method ofbringing integrin, collagen and the inhibitory compound, as described,or a cell comprising the same and the inhibitory compound. In vitro orex vivo this is achieved by exposing the integrin, collagen to theinhibitory compound or cells comprising the same to the compound orinhibitory agent in a suitable medium. For in vivo applications, anyknown method of administration is suitable as described herein.

The following example(s) are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion.

EXAMPLE 1

Recombinant I Domains

Recombinant I domains of integrin α1 and α₂ subunits were generated andisolated as (Xu, Y et al., 2000; Rich, R. L. et al., 1999). Purifiedrecombinant proteins were examined by SDS-polyacrylamide gelelectrophoresis (PAGE) followed by staining with Coomassie blue.

EXAMPLE 2

Purification of Recombinant Procollagen

Frozen yeast cells expressing recombinant type I and III procollagenswere obtained from FibroGen, Inc (San Francisco, Calif.). The yeastcells expressed both genes encoding human collagen and prolyl4-hydroxylase enabling formation of hydroxyproline residues andthermally stable triple helical collagen. The cells were thawed in anambient-temperature water bath and resuspended in a Start buffer (0.1 MTris, 0.4 M NaCl, 25 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF),1 mM pepstatin, pH 7.5). The cells were lysed using a French press, andthe lysate was centrifuged at 30,000×g for 30 min at 4° C. Thesupernatant was filtered through a 0.45-mm membrane, and the pH of thefiltrate was adjusted to 7.5. An affinity column was prepared bycoupling a recombinant collagen-binding MSCRAMM from Staphylococcusaureus (Patti et al., 1992) to CNBr-activated Sepharose 4B (AmershamBiosciences). The supernatant was applied to the column and incubatedovernight at 4° C. The column was washed with the Start buffer and boundmaterial was eluted with 0.5 M acetic acid. Fractions were examined bySDS-PAGE (4%/8%) under reducing conditions followed by Coomassie bluestaining. Fractions with procollagen were pooled. The concentration ofthe procollagen was estimated by comparing its band intensity with thatof known amount of type I collagen (Vitrogen) in Coomassie blue stainedSDS-gels.

EXAMPLE 3

Surface Plasmon Resonance (SPR) Measurements

For the analyses of interactions between recombinant I domains andfibrillar collagens, SPR measurements were carried out at ambienttemperature using the BIAcore 3000 system (BIAcore, Uppsala, Sweden) asdescribed previously (Rich et al., 1999) with following modifications.First, purified recombinant human procollagens I and III (describedabove), or bovine mature type II collagen (Sigma) were immobilized onthe flow cells of a CM5 chip resulting in 200-700 response units ofimmobilized protein. Different concentration of the α₁I and α₂I proteinsin HBS buffer (25 mM HEPES, 150 mM NaCl, pH 7.4) containing 5 mMβ-mercaptoethanol, 1 mM MgCl2, and 0.05% octyl-D-glucopyranoside werepassed over the immobilized collagen at 30 μl/min for 4 min.Regeneration of the collagen surfaces was achieved with 20 μl of HBScontaining 0.01% SDS.

Binding of α₁I and α₂I to a reference flow cell, which had beenactivated and deactivated without the coupling of collagen, was alsomeasured and subtracted from the response to collagen-coated flow cells.SPR sensorgrams from different injections were overlaid using theBIAevaluation software (BIAcore AB). Data from the steady state portionof the sensorgrams were used to determine the binding affinities. Basedon the correlation between the SPR response and change in protein masson the surfaces of flow cells, values for the binding ratio, ν_(bound),and the concentration of free protein, [P]_(free), were calculated usingthe equations described previously (Rich, R. L. et al., 1999). Scatchardanalysis was performed by plotting ν_(bound)/[P]_(free) againstν_(bound) in which the negative reciprocal of the slope is thedissociation constant, K_(D). Nonlinear regression was also performed byplotting ν_(bound) against [P]_(free) and fitted with the one-bindingclass or the two-binding class models using the GraphPad Prism™ software(GraphPad Software Inc., San Diego, Calif.). Results from the two modelswere compared with respect to the value of R² and the degree of freedomof the curve fit. The model that gave K_(D) values outside theexperimental data range was excluded. Experimental results werereproducible with at least three independent protein preparations.

SPR measurements for the analyses of the interactions between I domainsand synthetic collagen peptides were carried out at 15° C. using theBIAcore 3000 system. The synthetic collagen peptide was immobilized ontoa flow cell of a CM5 chip and various concentrations of recombinant Idomains were passed over the coated surface at 50 μl/min. Responses on areference flow cell were substracted from responses of thepeptide-coated flow cell. The BIAevaluation 3.0 software was used todetermine the association and dissociation rates (k_(on) and k_(off)),and K_(D) with a 1:1 binding model. R_(max), of fitting was similar tocalculated R_(max). The Chi² of each fitting was less than 2.

EXAMPLE 4

Competition Enzyme-linked Immunosorbent Assay (ELISA)

Microtiter wells (Immulon 4, Thermo Labsystems) were coated with 1 μg ofmature bovine type I collagen (Vitrogen) or purified human type IIIprocollagen in HBS for 2 hrs at room temperature. The wells were washedwith HBS and incubated with a blocking buffer (HBS containing 0.1% w/vovalbumin and 0.05% v/v Tween 20) overnight at 4° C. Varyingconcentrations of peptides were mixed with fixed concentrations ofrecombinant I domains in the blocking buffer containing 1 mM MgCl2 and 5mM β-mercaptoethanol and then added to the wells. After incubation at 4°C. for 3 hrs with gentle shaking, the wells were extensively washed withHBS containing 0.05% Tween 20 and 1 mM MgCl2. Bound α₁I or α₂I wasdetected by incubation with an anti-His monoclonal antibody (AmershamBioscience) diluted 1:3000 in the blocking buffer containing 1 mM MgCl2for 1 hr at room temperature, followed by incubation with goatanti-mouse IgG (H+L)-alkaline phosphatase conjugate (Bio-Rad) (1:3000dilution in the blocking buffer 1 mM MgCl2) for 1 hr at roomtemperature. Bound antibodies were quantified by adding 100 μl of 1.3 Mdiethanolamine, pH 9.8, containing 1 mM MgCl₂, and 1 mg/ml p-nitrophenylphosphate (Southern Biotechnology Associates, Birmingham, Ala.) to eachwell. The absorbance at 405 nm (A405 nm) was measured after 20-40 min ofincubation at room temperature. Background binding to the wells wasdetermined by incubating the I domains in wells that had been treatedwith blocking buffer alone. These values were subtracted from the valuesgenerated in the collagen-coated wells to determine collagen-specificbinding. Data were presented as the mean value±S.E. of A_(405 nm)(n=3).

EXAMPLE 5

Rotary Shadowing and Electron Microscopy

Rotary shadowing and electron microscopy of I domain-collagen complexeswere performed as described previously (Xu, Y et al., 2000). Eachbinding event was measured from the C-terminal end of type III collagen,that is from the base of the globular domain, and to the middle of thebinding spot. The binding events were then binned for every 10 nm alongthe collagen strand. The percentage of the number of events in each binover total events counted was calculated and plotted against the lengthof the collagen strand.

EXAMPLE 6

Synthesis and Purification of Collagen Peptides

Peptides were synthesized by a solid phase method on a TentaGel R RAMresin (RAPP Polymere GmbH, Tubingen, Germany) using Fmoc chemistry and amodel 396 MBS Multiple Peptide Synthesizer from Advanced ChemTech Inc.(Louisville, Ky.). Fmoc amino acids were purchased from Novabiochem, SanDiego, Calif. Coupling of amino acids was carried out twice usingdiisopropylcarbodiimide/1-hydroxybenzotriazole for 60 min. Fmocdeprotection was carried out using a mixture of 2% (v/v) piperidine and2% (v/v) 1,8-diazabicyclo-[5.4.0]undec-7-ene in dimethylformamidefollowed by treatment with 25% piperidine in dimethylformamide. Sidechains were protected with the following groups: t-butyl (Glu, Ser, andhydroxy-Pro), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Arg), and trityl(Gln).

After the completion of synthesis, peptide resins were washed thoroughlywith dimethylformamide, ethanol, and ether and then dried in a vacuumdesiccator. Peptides were released from the resin by treatment with amixture of trifluoroacetic acid, thioanisole, ethanedithiol, andtriethylsilane (90:5:2.5:2.5 by volume) for 8 h. The resins werefiltered, and the peptides were precipitated with cold anhydrous ether.The precipitate was washed with anhydrous ether three times and dried.The cleaved peptides were analyzed by reverse phase high pressure liquidchromatography on a Waters 625 liquid chromatography system (Milford,Mass.) using a Waters Delta-Pak C18 column.

EXAMPLE 7

Circular Dichroism (CD) Spectroscopy

Synthetic collagen peptides were analyzed by CD spectroscopy, asdescribed previously (Xu, Y. et al., 2000) with the followingmodifications. Briefly, peptides were dissolved in HBS to aconcentration of 50 μM. CD spectra were collected on a Jasco J720spectropolarimeter (Tokyo, Japan) from 190 to 240 nm, with bandwidth of1 nm and integrated for 1 s at 0.2 nm intervals. Samples were measuredat room temperature using cuvettes with 0.02 cm path length. Fortemperature-dependant denaturation analysis, 30 mM of peptides wereadded to a thermostatically controlled cuvette with a 0.5 cm pathlength. Thermal transition profiles were recorded at 225 nm as describedabove with a temperature slope of 20° C./hr. To calculate thetemperature melting points, the thermal transition profiles were fittedwith Boltzmann sigmoidal model using the GraphPad Prism™ software(GraphPad Software Inc., San Diego, Calif.).

EXAMPLE 8

Reagents and Cell Culture

The human recombinant mature type III collagen used for cell attachmentassays was purchased from FibroGen. All cell culture media componentswere purchased from Invitrogen. The human lung fibroblast cell lineMRC-5 was purchased from American Type Culture Collection (ATCC)(Manassas, Va.). The cells were cultured and passaged in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum,100 unit/ml penicillin and 100 μg/ml streptomycin. The cells were grownto subconfluence and passaged every 2-3 days.

EXAMPLE 9

Cell Attachment Assay

MRC-5 cells were starved overnight in serum deficient DMEM containingpenicillin and streptomycin, then detached using 1 mM EDTA and 0.025%trypsin at 37° C. for 2 minutes. The cells were washed with PBS andresuspended in DMEM containing 0.2% BSA supplemented with 2 mM MgCl2.100 μl of the cell suspension (˜1.5×10⁵ cells/ml) were added to themicrotiter wells coated with different concentrations of collagen orcollagen peptides and blocked with PBS containing 0.5% (w/v) BSA. Afterincubation at room temperature for 45 min, the wells were washed withPBS. Attached cells were fixed with 3% p-formaldehyde for 10 min at roomtemperature. Following washing with cold Tris-buffered saline (TBS), pH7.4, cells were fixed again in 20% methanol for 10 min and stained with0.5% crystal violet for 5 min. The wells were thoroughly washed withdistilled water and air-dried. Sodium citrate (0.1 M) was then added tothe wells to dissolve the dye and the absorbance at 590 nm was measured.The maximum cell attachment on type III collagen was set to 100% and,residual attachment on BSA was set to 0%.

EXAMPLE 10

Computer Modeling

The coordinates of the crystal structure of α₂I in complex with asynthetic collagen peptide were obtained from Protein Data Bank (code1dzi) and used as a template for the model studies. First, the Pheresidues in both the middle and trailing strands were replaced by Argresidues. Then local minimization was carried out in sizes of 5 Å forbest fit. Several basic components (i.e., hydrogen bond, van der Waalsand electrostatic interactions) contributing to the binding energybetween α₂I and the mutated collagen peptide were analyzed. Themolecular modeling experiment was carried out under ECEPP/3 force fieldby using the ICM software (Molsoft, La Jolla, Calif.).

EXAMPLE 11

Characterization of the Binding of α₁I and α₂I to Type I, II and IIICollagens

The interactions between the two I domains and fibrillar collagens(types I, II and III) were examined by SPR. Solutions of 1 μM α₁I or α₂Iwere passed over chips containing immobilized collagen I, II, or III inthe presence of 1 mM MgCl2. Both α₁I and α₂I showed binding to all threetypes of collagen (FIG. 1), consistent with previous reports (Nykvist, Pet al., 2000). To determine the dissociation constants (K_(D)) for theinteractions between the I domains and each collagen, increasingconcentrations of recombinant I domains (0.01-50 μM) were passed overthe collagen surfaces. In previous SPR studies using BIAcore 1000system, two classes of binding sites in type I collagen having differentaffinities for α₁I were shown (K_(D1)=0.26±0.01 mM, and K_(D2)=13.9±3.0mM), whereas α₂I appeared to have one class of binding sites (˜10 mM)(Rich et al., 1999).

The BIAcore 3000 system used in the present invention had highersensitivity of detection which enabled examination of the interactionsbetween α₂I and collagens at a sub-micromolar concentration range.Analyses using the SPR responses in the steady state portion of thesensorgrams, which indicates the equilibrium condition, showed that bothI domains have at least two classes of binding sites in the three typesof collagen. The dissociation constants (K_(D)) of these interactionsare summarized in Table 2. α₁I binds all three types of collagen withsimilar affinities (K_(D1)=˜0.15−0.32 mM, and K_(D2)=˜5.5−7.3 mM). Thebinding affinities of α₂I to the three types of collagen appeared to beslightly more variable. The K_(D) values for the high affinity bindingclass range from ˜0.3 μM for types I and III collagen, to 1.75 μM fortype II collagen, whereas the K_(D) values for the low affinity bindingclass range from ˜4 μM for type I collagen, to ˜16.5 μM and ˜14.5 μM fortype II and III collagen, respectively.

The two recombinant I domains also exhibited different binding kineticsto the collagens as indicated by the shape of the corresponding SPRsensorgrams (FIG. 1). Comparison of the shapes of the SPR sensorgrams ofα₁I with those of α₂I indicated a much slower association anddissociation rate of α₁I compared to α₂I in agreement with previousreports (Xu, Y et al., 2000; Rich, R. L. et al., 1999). However therewas no dramatic difference between each I domain binding to type I, IIand type III collagen. Thus, the binding characteristics of theinteractions between α₁I/α₂I and type III collagen are similar to thoseof the interactions between α₁I/α₂I and type I/II collagen.

TABLE 2 Summary of the binding affinities of α₁I and α₂I to fibrillarcollagens (types I–III) K_(D) ^(a) α₁I α₂I Collagen I 0.32 ± 0.10 0.26 ±0.08  5.5 ± 1.46 3.99 ± 0.82 Collagen II 0.15 ± 0.03 1.75 ± 0.09 7.28 ±1.16 16.5 ± 3.89 Collagen III 0.19 ± 0.03 0.33 ± 0.03 6.15 ± 0.95 14.5 ±3.41 ^(a)KD was calculated by equilibrium analysis. Data are presentedas mean value ± S.E of three independent studies.

EXAMPLE 12

Localization of a High Affinity α₁I and α₂I Binding Region in Type IIIProcollagen

Two sequence motifs, GFOGER and GLOGER, were identified as high affinitybinding sites in triple helical collagen for α₁I, α₂I and α₁₁I (Zhang,W. M. et al., 2003; Siljander, P. R. et al., 2004, Xu, Y et al., 2000).The fact that these sequences were present in type I and I collagen butnot in type III collagen suggested the presence of at least one novelhigh affinity binding site in type III collagen. To locate the highaffinity binding region(s) in type III collagen, collagen and I domaincomplexes were examined by rotary shadowing followed by electronmicroscopy (EM). Type III procollagen was used in these experimentssince it contains a globular-shaped C-terminal propeptide that allowsthe determination of the orientation of collagen molecules in EM.

Type III procollagen was incubated with α₁I or α₂I under bindingconditions and the complexes were then subjected to rotary shadowing andEM. The helical portion of the majority of the collagen molecules wasfound to be ˜300 nm long, indicating that these molecules were mostlyintact, full-length molecules. Multiple binding sites in the helicalportion of type III collagen were observed for both α₁I and α₂I (FIG.2), however, one region at 270-300 nm from the C-terminal end of themature chain contained approximately 75% and 25% of the total bindingevents of α₁I (n=269) and α₂I (n=299), respectively (FIG. 3), suggestingthat this region contained high affinity binding site(s) for α₁I andα₂I. Furthermore, adding EDTA to the incubation buffer before the rotaryshadowing dramatically reduced the number of I domains bound toprocollagens, suggesting that this binding was metal ion dependent (datanot shown).

EXAMPLE 13

Synthesis and Characterization of Collagen-Like Peptides MimickingPutative High Affinity Binding Sites in Type III Collagen

Type III collagen is a homotrimer composed of three α1(III)polypeptides, each containing 1029 amino acid residues in the maturechain (GenBank™ accession number P02461). Given that the averagecollagen molecule measured 300 nm, the average length per residue ofcollagen was 0.29 nm (3.43 amino acid residues/nm), which was consistentwith previous calculations (Bella et al., 1994). Based on thiscorrelation, the region located 270-300 nm region from C-terminal end ofthe mature chain corresponded to amino acid residues 168-270 of theα1(III) chain. This stretch of sequence contained one GER motif precededby GROGRO (SEQ ID NO: 13) and followed by GLO (FIG. 4 A). If a GERsequence was critical for integrin binding, this collagen sequence was apotential high affinity site for α₁I and α₂I. There was another GERmotif preceded by GAO and followed by GROGLO (SEQ ID NO: 14) close toC-terminal side of the 270-300 nm region. Therefore, peptides(GPO)₃GROGROGERGLO(GPO)₃ (peptide #1; SEQ ID NO: 15) and(GPO)₃GAOGERGROGLO(GPO)₃ (peptide #2; SEQ ID NO: 16) were synthesizedand used in I domain binding assays. Peptide (GPO)₁₁ (peptide #3; SEQ IDNO: 17), was used as a control peptide (FIG. 4B).

The synthetic peptides were examined for their ability to formcollagen-like triple helices by CD spectroscopy. The CD spectra of allfour peptides showed the characteristic ellipticity maxima at 220-225nm, indicating that they were capable of forming collagen-like triplehelices (FIG. 4C). The temperature-dependent unfolding of the triplehelix was followed by monitoring the CD at 225 nm. The reduction of themaxima was seen from about 35° C.-with melting points for the triplehelix structure of these peptides recorded between 41 and 44° C. (datafor peptide #1 are shown in FIG. 8B). The data discussed herein showedthat the peptides formed triple helix structure at temperatures (4-25°C.) used in the following experiments.

EXAMPLE 14

Inhibition of α₁I and α₂I Binding to Type I and Type III Collagen bySynthetic Collagen Peptides

To determine whether the type III collagen peptides contained highaffinity binding sites for α₁I and α₂I, their ability to inhibit thebinding of recombinant I domains to type I and III collagens wasexamined using ELISAs type assays. Various concentrations of peptides(0.01-100 μM) were incubated with recombinant I domains before themixtures were added to microtiter wells coated with type I or IIIcollagen. The results indicated that at 100 μM, peptide #1 (SEQ ID NO:15) inhibited the binding of α₁I and α₂I to type III collagen by 100%and 80%, respectively (FIGS. 5A and B). Peptide #2 (SEQ ID NO: 16) at100 μM inhibited the binding of α₁I and α₂I to type III collagen by 40%and 60% respectively, suggesting that although peptide #2 (SEQ ID NO:16) was recognized by the I domains, it was not a high affinity bindingsite. The control peptide #3 (SEQ ID NO: 17) did not show any inhibitoryactivity. The IC₅₀ values of peptide #1 (SEQ ID NO: 15) with α₁I and α₂Ibinding to type III collagen were 1.0±0.6 μM and 1.9±0.9 μM,respectively. Similar results were obtained with type I collagen; theIC₅₀ values of peptide #1 (SEQ ID NO: 15) with α₁I and α₂I binding totype I collagen were 1.4±0.4 μM and 0.14±0.09 μM, respectively (FIGS. 5Cand D).

EXAMPLE 15

Characterization of the Binding of I Domains to Collagen Peptide #1

The direct binding of the α₁I and α₂I domains to peptide #1 wasinvestigated further using SPR. Peptide #1 (SEQ ID NO: 15) wasimmobilized onto a CM5 chip. Increasing concentrations of I domains(0.5-30 nM) were passed over the surface containing peptide #1 (SEQ IDNO: 15). α₁I and α₂I exhibited similar association rates for peptide #1,5.6 Ms⁻¹×10⁴ and 4.4 Ms⁻¹×10⁴, respectively, however, α₁I showed a muchslower dissociate rate than α₂I, 1.3 s⁻¹×10³ compared to 12 s⁻¹×10³ forα₂I (FIGS. 6A and B, and Table 3). This resulted in a K_(D) of 23 nM forthe interactions between α₁I and peptide #1 (SEQ ID NO: 15), and a K_(D)of 283 nM for the interactions between α₂I and peptide #1 (SEQ ID NO:15) (Table 3). To test whether the binding of α₁I and α₂I to peptide #1(SEQ ID NO: 15) was metal ion dependent, 30 nM of each I domain in thepresence of either 1 mM MgCl2 or 2 mM EDTA was passed over a peptide #1(SEQ ID NO: 15)-coated surface. The presence of EDTA completelyabolished binding, indicating that the interactions were dependent onthe presence of divalent cations (FIG. 6C).

TABLE 3 Analyses of the binding of α₁I and α₂I to synthetic collagenpeptide #1 (SEQ ID NO: 15). k_(on)(MS⁻¹(×10⁴)) k_(off)(S⁻¹(×10⁻³))k_(D)(nM) α₁I 5.6 1.3 23 α₂I 4.4 12 283

EXAMPLE 16

Adhesion of MRC-5 Cells to Peptide #1 (SEQ ID NO: 15) Substrates

In order to investigate whether the synthetic collagen peptide #1 (SEQID NO: 15) was able to support cell adhesion, the human lung fibroblastcell line, MRC-5, which was shown previously to express comparablelevels of α₁β₁ and α₂β₁ integrins (Humtsoe et al., 2005) was used. Wellsin 96-well plates were coated with increasing concentrations of type IIIcollagen, peptide #1 (SEQ ID NO: 15) or #2 (SEQ ID NO: 16) and 1.5×10⁴MRC-5 cells were added to each well. The plate was incubated for 45 minat room temperature and adhering cells were quantified as describedherein. The results showed that peptide #1 (SEQ ID NO: 15) and type IIIcollagen could support adhesion of MRC-5 cells in a dose-dependentmanner, whereas peptide #2 (SEQ ID NO: 16) could not (FIG. 7).Considerable cell spreading was observed among cells incubated onpeptides #1 (SEQ ID NO: 15) at room temperature for 1.5 hrs similar tocells seeded on type III collagen (data not shown).

EXAMPLE 17

GROGER (SEQ ID NO: 4) is a Minimal α₁I/α₂I High Affinity Binding Motif

To determine the minimal binding sequence in peptide #1 (SEQ ID NO: 15),a shorter peptide containing the GROGER (SEQ ID NO: 4) sequence flankedby three GPO repeats at either ends was synthesized. Peptides containingGFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO: 2) motifs were also madefor comparison (FIG. 8A). All three peptide were able to form triplehelices as shown by their ellipticity maxima around 225 nm in the CDspectra (data not shown). In addition, all three showed a sharp decreasein their ellipticity at 225 nm as temperature increased. The meltingtemperature (Tm) of the GFOGER (SEQ ID NO: 1), GLOGER (SEQ ID NO: 2) andGROGER (SEQ ID NO: 4) peptides were determined to be between 37° C. and41° C. The denaturation profile of GROGER (SEQ ID NO: 4) peptide wascompared to that of the peptide #1 (SEQ ID NO: 15). The Tm of the GROGER(SEQ ID NO: 4) peptide was 41° C., slightly lower than the Tm of peptide#1 (SEQ ID NO: 15) which was determined to 43° C. (FIG. 8B).

To test whether GROGER (SEQ ID NO: 4) represented a high affinitybinding site for α₁I and α₂I, ability of the three peptides to inhibitthe binding of the two I domains was compared to type III collagen usingcompetition ELISAs. In these experiments, recombinant human mature typeIII collagen from FibroGen was used instead of procollagen III andpeptide #1 was used due to the limited availability of the later. Therecombinant I domains bound mature type III collagen in a similar way asto type III procollagen (data not shown). The results showed that GROGER(SEQ ID NO: 4), as well as GFOGER (SEQ ID NO: 1) and GLOGER (SEQ ID NO:2), inhibited the binding of α₁I and α₂I to the immobilized collagen(FIGS. 8C and D). The IC₅₀ values of GFOGER (SEQ ID NO: 1), GLOGER (SEQID NO: 2) and GROGER (SEQ ID NO: 4) were 1.9±0.03 μM, 2.2±0.01 μM and3.6±0.09 μM respectively and, for the inhibition of α₂I binding were1.4±0.1 μM, 12.0±1.1 μM and 1.1±0.04 μM, respectively. In addition, theIC₅₀ values determined for GROGER (SEQ ID NO: 4) were in the same rangeas those for the peptide #1, which was used as positive control in thisexperiment. Thus, GROGER (SEQ ID NO: 4) represented a minimal highaffinity binding sequence for α₁I and α₂I.

EXAMPLE 18

Molecular Modeling of the Interactions Between GROGER (SEQ ID NO: 4) andα₂I

The crystal structure of α₂I in complex with a synthetic collagenpeptide containing the sequence GFOGER (SEQ ID NO: 1) has been reported(Emsley, J. et al., 2000). The structure shows that the Glu residuedirectly interacted with the divalent cation Mg²⁺ co-ordinated by theMIDAS motif found in the α₂I. Another high affinity binding sitecomposed of the sequence GLOGER (SEQ ID NO: 2) also contains ahydrophobic residue at the second position. However, the presentinvention reported an integrin-binding sequence GROGER (SEQ ID NO: 4)that contained a charged Arg residue at the second position. In order toexamine how the α₂I structure accommodated this charged residue, inplace of a hydrophobic residue, computer modeling was performed. In thepublished α₂I-GFOGER (SEQ ID NO: 1) complex structure (PDB code 1dzi),Phe in the middle strand of the collagen triple helix participated invan der Waals interactions with the side chains of Asn¹⁵⁴ and Gln²¹⁵ ofα₂I and Phe in the trailing strand participated in van der Waalscontacts with Leu²⁸⁶ and Tyr¹⁵⁷ of α₂I (Emsley, J et al., 2000).Replacing Phe with Arg in the collagen peptide did not change thepositions of neighboring residues in α₂I. In the analysis of themolecular interactions between specific residues of α₂I and Arg in bothmiddle and trailing strands, the modified complex retained the van derWaals interactions previously described in the interaction with Phe(FIG. 9). Furthermore, a new hydrogen bond interaction was observedbetween the carbonyl backbone of Gln₂₁₅ of α₂I and the Arg residue inthe middle strand with a distance of 2.1 Å (FIG. 9, B). Thus, itappeared that the second position in the collagen peptide sequence wastolerant to substitutions, and that GROGER (SEQ ID NO: 4) represented anovel binding motif for the collagen-binding I domains.

EXAMPLE 19

Computer-Aided Molecular Modeling to Identify Integrin SpecificInhibitors

As discussed earlier, the interaction between integrin and collagen ismediated by the I domain present in the α chain of the integrin. Thecrystal structure of the α₂I domain in complex with a collagen triplehelix peptide showed that GFOGER (SEQ ID NO: 1) represented a bindingsite for α₂I in collagen. Similarly, GLOGER (SEQ ID NO: 2) wasrecognised by α₂I as well as by α₁I, α₁₁I.

Molecular modeling approach was used to define all binding sequences incollagen. It was observed that the integrin domains could bind to anumber of sites in collagen. It is contemplated that some of the sitesare recognized by all integrins whereas others are specificallyrecognized by individual I-domains (FIGS. 10A-D). Thus, these specificsequences provide a base for developing integrin specific inhibitors.

The following references were cited herein:

-   Bella, J et al., 1994, Science 266: 75-81.-   Camper, L., et al., 1998, J Biol Chem 273: 20383-20389-   Emsley J, et al., 2000, Cell 101: 47-56.-   Gelse, K. et al., 2003, Adv Drug Deliv Rev, 55: 1531-1546.-   Gullberg, D. E. and Lundgren-Akerlund, E., 2002, Prog Histochem    Cytochem 37: 3-54.-   Hulmes, D. J., 1992, Essays Biochem, 27: 49-67.-   Humtsoe, J. O., et al., 2005 J Biol Chem, 280: 13848-13857.-   Hynes, R. O., 1992, Cell, 69: 11-25.-   Knight, C. G., et al., 1998, J Biol Chem 273: 33287-33294-   Knight, C. G., et al. 2000, Journal of Biological Chemistry 275:    35-40-   Kramer, R. H. and Marks, N., 1989, J Biol Chem, 264: 4684-4688.-   Kuivaniemi, H. et al., 1997, Hum Mutat, 9: 300-315.-   Lee, J. O., et al. 1995, Structure 3, 1333-1340-   Liu, X., et al., 1997, Proc Natl Acad Sci USA 94: 1852-1856-   Myllyharju, J and Kivirikko, K. I., 2001, Ann Med, 33: 7-21.-   Nykvist, P., et al., 2000 J Biol Chem 275: 8255-8261.-   Patti, J. M. et al., 1992, J Biol Chem 267: 4766-4772.-   Prockop, D. J and Kivirikko, K. I., 1995, Annu Rev Biochem    64:403-434.-   Ramchandran, G. N. 1988, Int J Pept Protein Res., 31: 1-16.-   Rich, R. L., et al. 1999, J Biol Chem 274: 24906-24913-   Siljander, P. R., et al., 2000, Cell 101: 47-56.-   Velling, T. et al., 1999, J Biol Chem 274: 2573525742-   Xu, Y., et al., 2000, J Biol Chem 275: 38981-38989.-   Zhang, W. M., et al., 2003, J Biol Chem 278: 7270-7277

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually incorporated byreference.

One skilled in the art will appreciate readily that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those objects, ends and advantagesinherent herein. Changes therein and other uses which are encompassedwithin the spirit of the invention as defined by the scope of the claimswill occur to those skilled in the art.

1. A synthetic collagen-like peptide that is the sequence of SEQ ID NO:15; wherein amino acid Xaa in the sequence is hydroxyproline.
 2. Thesynthetic peptide of claim 1, wherein the peptide has a triple helicalstructure.
 3. The synthetic peptide of claim 1, wherein said peptidebinds I domains of integrins α1 and α2.
 4. The synthetic peptide ofclaim 1, wherein the peptides contain a binding motif forcollagen-binding I domains that is amino acid sequence SEQ ID NO:
 4. 5.The synthetic peptide of claim 4, wherein the motif is present on anN-terminus of human type III collagen or in the α1 and α2 chain of humantype 1 collagen.